JARQ 29, 201-205 (1995)

Pathological Studies on Natural and Experimental Porcine Pneumonia Caused by Porcine Reproductive and Respiratory Syndrome (PRRS) Virus

Masanori KUBO '1, Kumiko KII.IIIURA '1, Masaru KOBA Y ASHI'1, l.Vlitusgu SHI1VIIU'2, Shunji YAMADA'2, Tetsuo MOROZU1VII'3, Hideki KOBA Y AS1Il'3, Kenji lVIIT ANI'3, N obuyoshi ITO '3, Koshi YAMAMOTO'', Y asuo lVIIURA "2, Teruji YAMAMOTO''\ and Kazuo WATANABE'5

Abstract Porcine reproductive and respiratory syndrome (PRRS) was recorded in Chiba Prefecture in 1989. Twenty-four field cases were examined pathologically. The lungs showed consolidations grayish-pink in color in all the cases. Pericarditis was also observed. Histopathologically, the alveolar septa in the lungs were markedly thickened and alveolar spaces were narrow. In the cases with secondary bacterial infection, macrophages and neutrophils infiltrated the alveolar spaces. Lymphoid cells and macrophages also infiltrated the endo-and pericardium, and meninges. A virus was isolated from several field cases and identified as PRRS virus. The hysterectomy-produced colostrums-deprived (HPCD) piglets were inoculated with the virus. In the piglets sacrificed at 7 days after inoculation, interstitial pneumonia which was characterized by a thickening of the alveolar septa was observed. Severe lesions were observed at 28 days after inoculation, and they subsided 42 days after inocula-tion. The lesions in the piglets in which PRRS virus and Mycoplasma hyorhinis were inoculated, were more severe than those in the piglets in which only PRRS virus was inoculated. As a result of these observations, it was considered that Mycoplasma hyorhinis increased the severity of the lesions in the pig! ets infected with PRRS virus.

Discipline: Veterinary pathology Additional key words: arterivirus, mycoplasma

Introduction

Porcine reproductive and respiratory syndrome (PRRS) was first recognized in 1987 in the U.S.A. 2•3>. A similar syndrome was reported in Canada, Ger-many, the Netherlands, England, Belgium, and Spain. PRRS virus was first isolated in Lelystad, Netherlands8> in 1991. PRRS virus infection caused abortion in sow6>. Young pigs suffering from this disease showed pronounced hyperpnea, fever and in-terstitial pneumonia2•3>. This disease was popularly called Heko-Heko disease in Japan. Heko-Heko

disease has been recognized in Chiba Prefecture, Japan, since October 19897>. It occurred in large farms under poor sanitary conditions and 1 OOJo of the piglets died, although infertility and abortion were not observed. We examined these cases pathologi-cally, virologically and bacteriologically.

A virus isolated from Heko-Heko disease was designated as Chiba 92-1 5> and this virus was identi-fied as PRRS virus. We inoculated this virus into hysterectomy-produced colostrum-deprived (HPCD) piglets in order to reproduce the disease. We describe the results in this report.

• 1 Systemic Diagnosis Research Division, National Institute of Animal Health (NIAH)(Tsukuba, lbaraki , 305 Japan) • 2 Second Research Division, NIAH • 3 T hird Research Division, NIAH •

4 Planning and Coordination Division, NIAH • 5 Veterinary Clinical Training Center, Chiba Prefectural Federation for Agricultural Mutual Aid Associations

(Sahara, Chiba, 287 Japan)

202

Materials and methods

Field cases: Twenty-four piglets about 2 month-old which showed symptoms of Heko-Heko disease were sent to the National Institute of Animal Health from 2 farms in Chiba Prefecture for examination.

Bacteriological examinations: The general bac-teriological examinations were performed using blood agar plates. As for the isolation of Haemophilus spp., a special medium was used. Mycoplasma was isolated using mucin PPLO broth (M-broth).

Virological examinations: Sera and emulsion sam-ples prepared from the lungs, liver, spleen and mesen-teric lymph nodes were inoculated into the porcine kidney cell line cultures or alveolar macrophage cul-tures from HPCD pigs.

Experimental inoculation : HPCD piglets were maintained in stainless tubes covered with a plastic film. The piglets were fed recommended amounts of commercial milk substitute 2 or 3 times a day.

Four piglets were inoculated with 105 Chiba 92-1 strain of PRRS virus intranasally at 5 days of age. These piglets were euthanatized at 7, 14, 28, and 42 days postinoculation (pi.).

Two piglets were inoculated with Mycoplasma hyorhinis (Mhr) at 5 days after inoculation with PRRS virus at 21 days of age. One was moribund at JO days after inoculation with PRRS virus. The other was euthanatized at 26 days after inoculation with PRRS virus .

Two 26-day-old piglets were inoculated with Mhr. One of them was euthanatized for examinations at 21 days pi. and the other piglet was not necropsied

Plate I a. Marked thickening of alveolar septa in lung (field case)

Hcmatoxylin and eosin (HE) stain. ( X 100)

JARQ 29(3) 1995

because it did not show clinical signs. Histopathological examinations: At necropsy, the

organs from field and experimental cases were fixed in 10% phosphate-buffered formalin. These o rgans were embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin (HE).

lmmunohistochemistry: The Mhr antigen in the lung specimens from 6 field cases and one experimen-tal case, which was inoculated with both PRRS virus and Mhr, was examined using the avidu1 biotin com-plex method kit (Vectastain Ca., U.S.A.).

Results

Field cases: The lungs from 24 field cases showed consolidations grayish-pink i.n color, and sometimes fibrinous pleuritis. Pericarditis was found in 12 cases and peritonitis in one case.

Microscopically, interstitial pneumonia character-ized by a thickening of the alveolar septa was ob-served in 20 cases (Plate la). The thickening of the alveolar septa was mainly due to the proliferation of alveolar epithelial cells and infiltration of macro-phages (Plate I b). The macrophages infiltrated the alveolar spaces in all cases, and neutrophjls coexist-ed .in 12 cases. The cell infiltrations were pronounced in the case of bacterial infection. In 14 cases, there were perivascular and peribronchiolar infiltrations of plasma cells. Fibrinous pleuritis was seen in 5 cases. Macrophages and a few neutrophils infiltrated the pleura in these cases.

The Mhr antigen was detected on the surface of the bronchiolar epithelial cells .in 5 out of 6 cases examined.

Pla1e lb. High magnification of Plate la Proliferation of alveolar epithelial cells and macrophages. HE stain. ( x 400)

M. Kubo et al.: P(llhology of Porcine Pneumonia Caused b)' PRRS Virus 203

Plate 2. Heart in field case Macrophages and lymphocytes infiltrate the pericardium. There are fibrin deposits around the pericardium. HE stain. ( x 100)

Macrophage and lymphocyte infiltrations were seen in the pericardium, endocardium and occasion-ally in the myocardium (Plate 2). Mild nonpurulenL meningitis was observed in 14 cases, perivascular cuff-ing in 5 cases and accumulaLion of glial cells in .S cases. In the other major organs there were no microscopical lesions.

Bacteriologically, in most of the field cases exa-mined, 106-108 cfu/g of Mhr were isolated. Haemo-philus parasuis, Actinobaci/lus pleurop11eumo11iae and Pasteurella multocida were occasionally isolated.

Yirologically, when porcine kidney cell lines were used, reovirus and coronavirus were isolated from the lungs of 2 cases. Chlamydia were isolated from

Plate 3. Lung of piglet at 7 days after inoculation of porcine reproductive and respiratory syn-drome (PRRS) virus

Interstitial pneumonia is not severe and alveolar septa are thickened by infiltra-tion of macrophages, lymphocytes and de-generated cells. HE stain. ( x I 00)

the mesenteric lymph nodes from 4 out of 6 cases examined. PRRS virus was isolated from the sera and lungs of 5 out of 6 cases examined5>.

Experimental cases: The piglets which were in-oculated only with PRRS virus or Mhr did not ex-hibit gross lesions. Fibrinous pleuritis was observed in the piglet inoculated with PRRS virus and Mhr.

Microscopically, interstitial pneumonia was charac-teristic in the lungs of the piglets inoculated with PRRS virus .

In a piglet which was euthanatized at 7 days pi., interstitial pneumonia was sparsely distributed, but it was not severe (Plate 3). Alveolar septa were thick-ened due to the infiltration of macrophages and lym-phocytes. Small accumulations of degenerated cells with pyknotic nuclei were present in the alveolar septa.

In a piglet euthanatized at 14 days pi., the lesions of interstitial pneumonia became more severe and spread over larger areas of the lungs (Plate 4). The marked thickening of the alveolar septa was due to macrophage and lymphocyte .infiltrations.

The piglet which was euthanatized at 28 days pi., showed the most severe lung lesions. The alveolar septa were thickened by proliferation of alveolar epithelial cells. Plasma cell infiltration was seen around the peribronchiolar and perivascular areas in the lungs. Macrophages and lymphocytes infiltrat-ed the pericardium and endocardium. Mild menin-gitis was also seen.

Although the lesions in a piglet at 42 days pi. were milder, the cell components in the lesions were

Plate 4. Lung of piglet at 14 days after inoculation of PRRS virus

Alveolar septa are markedly thickened by infiltration of macrophages and lympho-cytes. HE stain. ( x 100)

204

the same as those in the piglet at 28 days pi. One of two piglets which were inoculated with

both PRRS virus and Mhr was moribund at 10 days pi. of PRRS virus, and showed the most severe lung lesions among the experimental cases. Alveolar epithelial cells proliferated and macrophages infiltrat-ed the alveolar septa and alveolar spaces (Plate 5). Cellular debris accumulated in the alveolar septa. Mhr antigen was detected on the surface of the bron-chiole by immunohistochemistry. The other pigleL which was euthanatized at 26 days after inoculation with PRRS virus, exhibited milder lesions with the same cell components as in the former case. Plasma

Plate 5. Lung of piglet at IO days after inoculation of PRRS virus and 5 days after inoculation with Mycoplasma hyorhinis (Mhr)

Alveolar septa are markedly thickened by the proliferation of a lveolar epithelium and infil tration of macrophages. Fibrin deposi ts in pleura. HE stain. ( x 100)

Plate 6. Heart of the same case as in Plate 5 Plasma cells and macrophages in filtrate the endocardium. HE stain. ( x 200)

JARQ 29(3) 1995

cells infiltrated the peribronchiolar areas in the lungs. Pleuritis, pericarditis and endocarditis (Plate 6) as well as meningitis (Plate 7) were also observed (Table I).

In the cases which were inoculated with Mhr alone, there were no recognizable lesions.

Discussion

It was reported that PRRS is characterized by interstitial pneumonia in young pigs1> and abortion in sows6>. In the herd in which Heko-Heko disease occurred, neither abortion nor reproductive disord-ers were observed by farmers. The major clinical signs consisted of laborious abdominal respiration in the piglets7>. The PRRS virus was isolated from the lungs and sera of the diseased piglets and it was designated as Chiba 92-1 S>. Since the inoculation of this virus induced interstitial pneumonia, as in the field cases, it was assumed that PRRS virus was the causal agent of Heko-Heko disease.

Histologically, interstitial pneumonia was charac-terized by the thickening of the alveolar septa and plasma cell infiltration in perivascular and peribron-chiolar areas. These lesions were commonly observed in the field cases. In experimental cases, slight inter-stitial pneumonia was already observed at 7 days pi. Up to 14 days pi. the thickening of the alveolar septa was mainly due to the infiltration of macro-phages. Thereafter, macrophages in the alveolar septa were gradually replaced by a proliferation of alveo-lar epithelial cells. The lung lesions showed the most severe appearance at 28 days pi. and the lesions at 42 days pi. became milder in appearance. Therefore,

-.

\

. .

Pla1e 7. Brain of piglet at 26 days after inoculation with PRRS virus and 21 days after inocula-tion with Mhr

Lymphocytes and macrophages aggregate in the meninges. HE stain. ( x 200)

M. Kubo et al.: Pathology of Porcine Pneumonia Caused l;y PRRS Virus 205

Table 1. Palhological lesions in field cases and cxperimenial cases

Field cases Expcrimcn1al cases PRRSV•> PRRSV + Mhr•l

7 14 2 1 42 de) 10 26 dd)

Alveolar sepia Thickening 20•>124r) + + + + + + Cellular debris 0/24 + + + +

Alveolar space Macrophages 24/24 + + + Ncutrophils 12/12 +

Pcribronchiolc Plasma cells 14/24 + + + +

Plcuritis 5/24 + + Hean

Carditis 14/24 + + + + Brain

Meningitis 14/24 + + + + Cuffing 5/24 + + + Glial nodules 5124 +

a): Porcine reproductive and respiratory syndrome virus, b): Mycoplasma hyorhinis, c): Days after inoculation, d): Days after inoculation of PRRS virus (Mhr was inoculated at 5 days after PRRS virus inoculation.), e): Number of positive pigs, f): Number of pigs examined. + : Lesions arc present, - · Lesions are absem.

il appears that interstitial pneumonia caused only by PRRS vi rus could be cured naturally, if there

is no secondary inrection with mycoplasma or bacteria.

Mild nonpurulent meningitis and peri- and endo-carditis were often observed in the field cases. These lesions were reported by other authors1•4 •9> and om:

experiments also confirmed t he same findings . It was considered that the PRRS virus had an affinity

not only to the lung tissues but also to the heart

tissues and meninges. Mhr was isolated from the lungs in most of the

field cases. However, the piglets inoculated only with

Mhr did not exhibit any lesions in the lungs. On the other hand, the pigs inoculated with both PRRS

virus and Mhr showed more severe lesions than those inoculated with PRRS virus alone. These results sug-gest that Mhr was not the causal agent of interstitial

pneumonia. However, Mhr may produce severe le-sions in the piglets which have been infected with

PRRS virus.

References

I) Collins, J.E. et al. (1992): Isolation of swine infer-tility and respiratory syndrome virus (isolate A TCC VR-2332) in North America and experimental

reproduction of the disease in gnotobiotic pigs. J. Vet. Diagn. Invest. , 4, 117- 126.

2) Loula, T. (1991): Mystery pig disease. Agri-Practice, 12, 23-34.

3) Morin, M. e1 al. (1990): Severe proliferative and necrotizing pneumonia in pigs: a newly recognized dis-ease. Can. Vet. J., 3 1, 837-839.

4) Pol, J.M. A. et al. (1991): Pathological, ultramuc-wral, and immuno-histochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease. Vet. Q., 13, 137-143.

5) Shimizu, M. er al. (1993): Isolation of porcine reproductive and respiratory syndrome (PRRS) virus from Heko-Heko diease of pigs. J. Ver. Med. Sci., 56, 389-391.

6) Terpstra, C., Wensvoort, G. & Pol, J.M. A. (1991): Experimental reproduction of porcine epidemic abor-tion and respiratory syndrome (mystery swine disease) by infection with Lelystnd virus: Koch's postulates fulfilled. Vet. Q., 13, 131-136.

7) Watanabe, K. (1992): Heko-Heko disease. Proc. Jpn. Pig Soc., 20, 15-16 (In Japanese].

8) Wensvoort, G. et al. (1991) : Mystery swine disease in the Netherlands: the isolation of Lelystad virus. Vet. Q., 13, 121-130.

9) Wcnsvoort, G. ct al. (1992): Lelystad virus, the cause of porcine epidemic abortion and respiratory syn-drome: a review of mystery swine disease research at Lelystad. Ver. Microbiol., 33, 185-193.

(Received for publication, Nov. 24, 1994)

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